Breast cancer is the most common female cancer worldwide. Genetic defects clearly play a role in breast cancer etiology; however, epidemiological studies suggest a strong effect of the diet or components of the diet on the development and progression of breast cancer. The consumption of milk has been negatively correlated to the incidence of breast cancer. Sphingolipids are especially rich in milk and milk products. Sphingolipids regulate signaling pathways that have been shown to be dysregulated in cancer, mediating cell growth, differentiation, and cell death. Although the sphingolipid metabolites ceramide and sphingosine are toxic to cells at higher concentrations, no toxic side effects of even large amounts of dietary complex sphingolipids have been reported. This may be due to the limited digestion. It is these properties that make them ideal candidates for chemopreventive agents. We have shown earlier that dietary sphingolipids suppress early and late stages of chemically induced colon cancer. However, small amounts of the sphingolipid metabolites are transported into the body, and this may be sufficient to suppress breast tumor growth as suggested by epidemiological data. In the studies under this application, we will use cell models for early and late stages of breast cancer, i.e., MCF10A, MCF10AT1 and DCIS cells, to characterize the response of normal, untransformed breast epithelial cells to exogenous sphingolipids. A comparison of the response of transformed breast epithelial cells will identify changes in signaling pathways mediating cell growth that may be responsible for the higher sensitivity of transformed cells to sphingolipids. Moreover, we will evaluate if non-toxic concentrations of sphingolipid metabolites can enhance the effects of conventional chemopreventive and chemotherapeutic agents. These studies will establish markers for sphingolipid efficacy in suppression of breast tumor growth that can be used in future in vivo studies. We also will be able to determine which combination of sphingolipid metabolites and chemotherapeutic agents is most promising in reducing cell growth and/or inducing apoptosis in tumor cells but do not affect normal cells. The results from these studies will provide the tools to conduct in vivo studies, and hopefully will lead to the development a concept of breast cancer chemoprevention with dietary sphingolipids as a non-toxic alternative to conventional treatment.